1. Field of the Invention
The present invention relates to the field of high performance liquid chromatography which determines the composition of a liquid medium by means of its refractive index.
The invention also relates to a process for measuring the difference between the refractive indexes of two media traversed by a light beam.
More precisely, the invention relates to a phase modulation and an interference fringe differential refractometer.
The use of the present invention allows improvements in high performance liquid chromatographs, known as HPLC, to be achieved.
2. Description of the Prior Art
Most detectors in use in the in this field are photometers and refractometers but also for very specific applications, electrochemical, conductometric or other detectors may be used.
Photometers have the advantage of exhibiting a high sensitivity and a great stability. They may be used in the case in which the eluates to be detected absorb the light within the range of wavelengths between about 190 and 700 .eta.m. But these photometers have major drawbacks: they are not universal devices and, in a single analysis, there may be eluates which absorb the light at different wavelengths or which even practically do not absorb it, which has the disadvantage, notably in preparative chromatography, of letting impurities pass unnoticed.
Furthermore, in the case of preparative chromatography, the photometer is rapidly saturated at around an optical density of the order of 2.
By dividing for example by 10 the length of the optical path in the vessel, this drawback is decreased but still more sensitivity is lost.
The main advantage of refractometers is that they are practically universal. Most of the devices which are currently manufactured use a light beam deviated through a double prismatic circulating vessel. In these detectors, a light source casts a beam onto a double photodetector after passing successively through a diaphragm, possibly concentrating lenses, a rotating glass strip with parallel faces for adjusting the optical zero of the device, i.e. for balancing the light intensity lighting up the two photodetectors, a double prismatic vessel, one for the reference liquid and the other for the phase to be analyzed.
When the refractive index of the phase to be analyzed varies, the prismatic section of the two successive vessels is such that the beam deviates from one photodetector to the other, according to the sign of difference of the indexes between the two vessels. However, in some cases, notably in preparative chromatography, where high concentrations may be found in one of the two vessels, or if an elution gradient is created, the index variations may be such that the beam may deviate to the point of saturation of the device, which means that the deviated beam eventually only lights up one of the two cells. The chromatogram is thus clipped and several peaks with a common base may no longer be distinguished.
As it is the case for photometers, these drawbacks may be decreased by reducing the deviations, but here again at the expense of a sensitivity loss. Saturation is then avoided, but the smaller peaks, i.e. impurities, are no longer distinguished in preparative chromatography.
Whatever the effort the problem created by an elution gradient is always present.
A refractometric system with a monochromatic source has also been proposed, whose beam is divided to pass through two vessels in parallel, one containing a reference liquid and the other, the phase to be analyzed, then the two beams are reassembled to light up a photodetector. Interferences occur because of the variation of the optical path as a function of the index variation. One may consider that the sinusoid followed by the intensity is linear in the vicinity of the zero difference index, which gives an acceptable sensitivity but does not solve the problems of saturation and of use of a gradient, and, as in the previous cases, saturation is avoided to the detriment of sensitivity.
French patent FR-2,596,526 illustrates an example of a refractometric detector in which each of the vessels (reference and measuring vessel) operative independantly in an interferometry system with the two vessels being supplied with light through the same source.
According to this prior art, the photometric detection is performed by two independent photodetectors which each receive a light intensity which is a sinusoidal function of the difference of the refractive indexes between the reference vessel or air and the measuring vessel. An individual calibration of each of the photometers is thus necessary.
This optical system notably includes a piezoelectric element for vibrating a mirror in which part of the light beam coming from the source is reflected. However, the piezoelectric element may generate a certain measurement drift (linked to the temperature rise) in time. Besides, the motion of the mirror by the piezoelectric element is necessarily limited in frequency and/or in amplitude, which allows neither a wide measuring range nor a high measuring accuracy.
It is well-known, as disclosed in French patent 2,254,996, as disclosed in patent application DT 25 18 197 or as disclosed in U.S. Pat. No. 4,289,403, that the measurement of an optical path may simply be extracted during the measuring of the phase shift recorded between the signal delivered by a photodetector located in an interference figure and the modulation applied to only one of the beams of the interferometer. This measurement of the optical path gives, for a given test distance, the refractive index difference for a stable reference or, when the index is known and remains constant, the same measurement allows the test distance to be determined.
These various teachings agree that the interferometric device must be stable, as compact as possible, and symmetrical from the viewpoint of the path taken by each of the beams.
U.S. Pat. No. 4,289,403 confirms this observation since the object there of is a modulator of a new kind which minimizes the optical path difference for each of the two beams passing therethrough, even if the resulting modulation exhibits some non-linearities.
In the German Patent 2,518,197, a compensation applied to the modulator allows the phase shift amplitude to be obtained. This system provides a fast response in the analysis of the phase shifts generated by variations in a physical quantity. This patent clearly shows that a compensation, in terms of high voltage for a Pockels cell for example, applied to the modulator is no longer linear over a phase shift of the order of 2.times.180.degree. (approximately 4.lambda.) and that a compensation higher than this value would damage the modulator. The dynamics of the device is thus limited thereby.